Variants of PAI-2

ABSTRACT

Variants of the plasminogen activator inhibitor PAI-2 in which the 66-98 amino acid residue region has been altered to eliminate at least one protease sensitive site are provided. The variants of the invention maintain the biological activity of PAI-2 and amino acids up to 65 and from 99 of PAI-2 in frame. The PAI-2 variants of the invention in labelled form, as well as DNA molecules encoding the variants of the invention, transformed host cells expressing the variants of the invention compositions and diagnostic kits comprising the variants of the invention, antibodies against the variants of the invention and processes for the production of the variants, DNA molecules, transformed hosts, compositions and antibodies of the invention are also described.

TECHNICAL FIELD

This invention relates to genetically engineered variants of aplasminogen activator inhibitor, PAI-2.

DEPOSITION OF MICROORGANISMS

E. coli strain BTA 1445 was deposited with the American Type CultureCollection of 12301 Parklawn Drive, Rockville Md. 20852, U.S.A. inaccordance with the provisions of the Budapest Treaty under accessionnumber ATCC 53585 on Feb. 11, 1987.

BACKGROUND ART

Plasminogen activators (PAs) are serine proteases which convert theabundant extracellular zymogen, plasminogen, into plasmin, an activeprotease which can promote degradation of all components of theextracellular matrix. (Dano et al. Adv. Cancer Res. 44: 139-266, 1985).

Two different types of PAs have been recognised in mammalian tissues:

(1) Tissue-type Plasminogen Activator (t-PA). t-PA is a serine proteasewith a molecular weight of about 70,000, composed of one polypeptidechain containing 527 amino acids. Upon limited digestion with plasminthe molecule is converted to a two-chain activator linked by onedisulphide bond. This occurs by cleavage of the Arg 275 - Ile 276peptide bond yielding a heavy chain (M_(r) 38,000) derived from theN-terminal part of the molecule and a light chain (M_(r) 32,000)comprising the COOH-terminal region. The catalytic site located in thelight chain of t-PA is composed of His 322, Asp 371 and Ser 478. t-PAspecifically catalyses the hydrolysis of an Arg 560 - Val 561 bond inplasminogen. Fibrin has been found to strongly stimulate plasminogenactivation by t-PA.

(ii) Urokinase-type Plasminogen Activator (U-PA). u-PA has an M_(r) of50,000 and occurs in a one-polypeptide and a two polypeptide chain form.The one chain form is an inactive proenzyme, while the two-chain form isthe active enzyme. u-PA has a substantial plasminogen activator activityin the absence of fibrin and is not stimulated by its presence. t-PA'shigh affinity for fibrin suggests that it is mostly associated with afibrinolytic function while u-PA is associated with extracellularproteolytic events such as tissue remodelling and destruction (i.e.organ involution, inflammatory reactions and particularly in theinvasive growth and metastatic spread of malignant tumours).

Experimental use of t-PA and single chain u-PA as thrombolytic agents inman has been promising. However, it has become apparent that PAs mayhave a less pronounced fibrin specificity in man than was anticipatedfrom several animal models, suggesting a need for further improvementeither of the agents or of their administrative schemes in clinicalthrombolytic therapy. One possibility is the use of specific fast-actingprotein inhibitors of PAs to modulate the systemic fibrinolytic effectsof PAs.

Recent evidence suggests that urokinase-mediated plasminogen activationmay also play a role in the invasive behaviour of malignant cells. Withfew exceptions malignant cells release PAs in abnormally high amounts.Ossowski and Reich (Cell 35: 611-619, 1983) reported that anti-urokinaseantibodies inhibited the metastasis of human epidermoid carcinoma cellsseeded onto chick embryo chorioallantoic membranes. Bergman et al (Proc.Natl. Acad. Sci. 83: 996-1000, 1986) have shown that protease nexin I, afibroblast-secreted inhibitor of urokinase and plasmin, effectivelyinhibits the cell mediated degradation of extracellular matrix (ECM) byhuman fibrosarcoma (HT1080) cells. Finally, Sullivan and Quigley (Cell45: 905-915, 1986) have demonstrated that a monoclonal antibody to PAinhibits the degradation of ECM by Rous sarcoma virus-transformed chickfibroblasts. It follows from these observations and those of others[e.g. Mignatti et al., Cell 47: 487 (1986); Ossowski, Cell 52: 321(1988); Reich et al, Cancer Res. 48: 3307 (1988)] that specific proteaseinhibitors of urokinase may play a critical role in altering the levelsof active tumour cell PA in tumour tissue and therefore influence tumourgrowth and invasion in vivo.

There are other indications that a specific inhibitor of urokinase-typeplasminogen activator has a role in modern medicine. PAs are involved ina range of inflammatory conditions such as arthritis. Plasmin candegrade cartilage [Lack, CH & Rogers, HJ (1958) Nature 182: 948] and lowlevels of fibrinolytic activity due to plasmin have been detectedhistochemically in synovial membranes. The PA/plasmin system has beendetected in rheumatoid cell cultures [Werb, Z et al. (1977) New Engl JMed 296, 1017] and elevated levels of uPA have been noted in rheumatoidsynovial fluid [Mochan, E. & Uhl, J. (1984) J. Rheumatol 11, 123].Hence, the use of a specific inhibitor of uPA in arthritis could reversethe tissue destruction associated with this disease.

Other conditions where the application of a specific PA inhibitor may beof use include diseases or conditions such as osteoarthritis, multiplesclerosis, colitis ulcerosa, SLE-like disease, psoriasis, pemphigus,corneal ulcer, gastroduodenal ulcer, purpura, periodontitis, haemorrhageand muscular dystrophy. Finally, a PA inhibitor could have a significantrole in skin wound healing and tissue repair especially since twotrypsin inhibitors have been shown to enhance formation of connectivetissue with increased tensile strength of the wound tissue [Kwaan, HCand Astrup, T (1969) Exp. Molec. Path. 11, 82] and keratinocytes areknown to produce both uPA and tPA [Grondahl-Hansen, J et al. (1988) J.Invest Dermatol].

PA inhibitors, members of the serpin gene family (Sprengers and Kluft,Blood 69: 381-387, 1987), have been classified into four immunologicallydifferent groups:

1) Endothelial cell type inhibitor, PAI-1.

2) Placental type PA-inhibitor, PAI-2.

3) Urinary type PA-inhibitor, PAI-3.

4) Protease Nexin I, PNI.

PAI-2 (M_(r) about 46,000) has been purified from placental tissue,monocytes and the human monocytic cell line U937. The PAI-2 inhibitorsfrom these different sources are immunologically related and recent cDNAsequence analyses of PAI-2 derived from human placenta and the humanU937 cell line confirmed they are identical, although two forms of themolecule exist differing in only 3 single amino acid residues. Both cDNAforms have been isolated from U937 cells. (Schleuning et al. Mol. Cell.Biol. 7: 4564-4567, 1987; Antalis et al. Proc. Natl. Acad. Sci. 85:985-989, 1988). PAI-2 reacts with both u-PA and t-PA (better with twochain t-PA than with single chain t-PA) to form SDS stable complexes.PAI-2 does not bind to fibrin or to fibrin-bound t-PA.

As is the case with most potent biologically active proteins, PAI-2 isproduced in very small amounts in vivo and as such is difficult topurify and characterise by conventional biochemical approaches. Therecent expression of PAI-2 in bacterial cells (Antalis et al. Proc.Natl. Acad. Sci. 85: 985-989, 1988; Bunn et al, Abstracts of the SecondInternational Workshop on the Molec. and Cell. Biol. of PlasminogenActivation, Brookhaven National Lab., April 1989), now allows theproduction of quantities of purified PAI-2 needed to evaluate itsbiological efficacy in the various potential clinical applicationsdescribed above.

DISCLOSURE OF THE INVENTION

Knowledge of the complete nucleotide sequence of PAI-2 allows specificgenetic manipulations to be made which produce variants of PAI-2 whichmay exhibit improved properties compared with the native molecule.

Desirable improved properties include increased in vivo half life,increased or altered specificity, and/or improved pharmaceuticaleffectiveness.

The alterations may provide different properties which open up new areasof application or variants which are more amenable to industrialproduction, thus leading to improved production processes.

A unique difference between PAI-2 and the other serpins is theadditional stretch of 33 residues (66-98) in the C₁ -D interhelicalregion [Huber, R and Carrell RW (1989) Biochemistry 28 8951-8966]. Thisregion is generally either limited to a short 9-residue stretch, as inovalbumin or is absent, as in other members of the superfamily (e.g.human αl-antitrypsin, human antithrombin III). The significance of thisstructure is unknown. The present inventors have discovered that thisregion is sensitive to proteases, leading to the generation of a 37 kDform of PAI-2 during production. The presence of a 37 kD contaminant inPAI-2 preparations is not likely to be acceptable to regulatoryauthorities. Further, the 37 kD form of PAI-2 is unable to bind to U-PA.

Thus it is desirable to provide biologically active PAI-2 moleculeswhich are not sensitive to protease. When providing a variant of aparticular protein which lacks an undesirable characteristic, it is notpossible to predict whether the variant will maintain the desiredbiological activity of the parent protein, particularly where thealterations are significant. Given that the 66-98 amino acid region ofPAI-2 is unique to PAI-2 it would be anticipated that alterations tothis region of the molecule would be likely to render the resultantmolecule inactive or at least have an adverse effect on its activity.Surprisingly, the variants of the present invention do retain thebiological activity of native PAI-2, whilst lacking proteasesensitivity.

Changes to PAI-2, can be made by modifying individual amino acids ofPAI-2 by site-directed mutagenesis of the DNA or by wholesalerestructuring by DNA deletion or insertion to provide variants of theinvention. The actual manipulations of the DNA can in general beperformed in accordance with standard techniques in the art. Thespecific changes exemplified are produced by restructuring by DNAdeletion.

According to a first embodiment of this invention there is provided aPAI-2 variant in which the 66-98 amino acid residue region of PAI-2 hasbeen altered to eliminate at least one protease sensitive site whichvariant maintains biological activity of PAI-2 and amino acids to 65 andfrom 99 of PAI-2 in frame. Preferably the variant is a deletion variant.

The invention particularly provides the PAI-2 variant Δ66-98 as hereindefined wherein Δ66-98 has amino acids 66-98 inclusive of the PAI-2amino acid sequence (SEQ ID NO: 1) deleted. The invention alsoparticularly provides the variant Δ74-96 as herein defined, whereinΔ74-96 has amino acids 74-96 inclusive of the PAI-2 amino acid sequencedeleted.

According to a second embodiment of this invention, there is provided aPAI-2 variant of the first embodiment in labelled form.

According to a third embodiment of this invention there is provided aDNA molecule, the sequence of which encodes a PAI-2 variant of the firstembodiment.

According to a fourth embodiment of this invention there is provided arecombinant DNA molecule comprising a DNA molecule of the thirdembodiment, and vector DNA.

Typically, the vector DNA is plasmid DNA.

Preferred plasmid vectors of the invention include E. coli expressionvectors such as those based on the P_(L) promoter, lac promoter, tacpromoter or trp promoter, pGEM4Z and vectors derived therefrom, pSp70and vectors derived therefrom, baculovirus transfer vectors such aspac373, pac360 and vectors derived therefrom, mammalian expressionvectors such as pBPV-1, pBPV-BV1, pdBPV-MMTneo, SV40 based expressionvectors such as pBTA613, and vectors derived therefrom, vaccinia virusexpression vectors, retroviral expression vectors and other vectors usedfor the expression of recombinant DNA molecules in homologous orheterologous hosts.

Vectors derived from these vectors are those vectors obtained by makingstructural alterations to these vectors. Examples of the types ofalteration include those made for the purpose of increasing expressionfrom a particular vector.

pBTA613 is a mammalian cell expression vector. Foreign genes areexpressed by cloning into the multiple cloning site flanked upstream bythe SV40 early promoter and downstream by SV40 polyadenylation signals.pBTA613 comprises the following fragments in order. The 345bpPvuII-HindIII fragment from the SV40 origin, 51bp HindIII-EcoRI multiplecloning sites from pUC18, 75bp EcoRI-AatII fragment from pBR327, 853bpBamHI-XhoI fragment from pMSG with AatII linkers attached to both ends,2262bp AatII-EagI fragment from pBR327, 27bp oligonucleotide(GGCCCATATGATATCTCGAGACTAGTC: SEQ ID NO: 4), 288bp EagI-SalI fragmentfrom pBR327, 345bp PvuII-HindIII fragment from the SV40 origin, 734bpHindIII-BglII fragment encoding mouse dihydrofolate reductase frompSV2-DHFR, 141bp Sau3A fragment from SV40 small t intron region and293bp Sau3A fragment from SV40 early polyadenylation region. The HindIIIsite at the 5' end of the dhfr gene was deleted using S1 nuclease, otherincompatible ends were made flush using S1 nuclease or filled in withdNTPs and DNA polymerase I (Klenow).

Preferred recombinant DNA molecules of the invention include pBTA829,pBTA840, pMINDEL 74-96, and derivatives of these recombinant DNAmolecules.

Derivatives of these recombinant DNA molecules are molecules derivedfrom these molecules and include molecules where alterations have beenmade to the DNA structure for purposes such as improving or altering thecontrol of expression of the encoded PAI-2 variant. The recombinant DNAmolecule derivatives of the invention maintain the PAI-2 variant codingregion of the parent molecule.

According to a fifth embodiment of this invention there is provided atransformed host cell transformed by a recombinant DNA molecule of thefourth embodiment.

Typically, host cell lines are derived from suitable E. coli K-12strains. They can also be derived from eukaryotic organisms, and caninclude COS cells, CHO cells, U937 cells, BHK-21 cells, Vero cells, CV1cells, C127 cells and cell lines derived from the insects Spodopterafrugiperda and Bombyx mori.

According to a sixth embodiment of this invention there is provided aprocess for producing a PAI-2 variant of the first embodiment, whichprocess comprises: deleting nucleotides from the 66-98 amino acidresidue region of a DNA molecule encoding PAI-2 such that the aminoacids up to 65 and from 99 remain in frame and the resulting variantmaintains the biological activity of PAI-2.

According to a seventh embodiment of this invention there is provided aprocess for producing a recombinant DNA molecule of the fourthembodiment, which process comprises inserting a DNA molecule of thethird embodiment into vector DNA.

According to an eighth embodiment of this invention there is provided aprocess for producing a transformed host cell of the fifth embodiment,which process comprises making a suitable host cell competent fortransformation, and transforming the competent host cell with arecombinant DNA molecule of the fourth embodiment.

According to a ninth embodiment of this invention there is provided atherapeutic and/or a diagnostic composition comprising an effectiveamount of at least one PAI-2 variant of the first embodiment togetherwith a pharmaceutically acceptable carrier, excipient and/or diluent.The pharmaceutically acceptable carriers, diluents and excipients whichmay be used can be selected from those standardly used in thepreparation of pharmaceutical formulations. When used for diagnosticpurposes the agent may comprise the at least one variant in labelledform. PAI-2 variants may be labelled with a radioisotope such as I¹³¹ orconjugated to an appropriate enzyme or other chemical agent.Particularly provided are such agents wherein the at least one variantcomprises Δ66-98 and/or Δ74-96, as herein defined. When used for theproduction of antibodies the composition may comprise an adjuvant.

According to a tenth embodiment of this invention there is provided amethod of inhibiting tumour invasion and/or treating tumours comprisingadministering to a patient requiring such treatment a therapeuticallyeffective amount of a PAI-2 variant of the first embodiment and/or acomposition of the ninth embodiment.

According to an eleventh embodiment of this invention there is provideda method of treatment of an inflammatory disease such as rheumatoidarthritis, osteoarthritis, inflammatory bowel disease, ulcerativecolitis, psoriasis or pemphigus comprising administering to a patientrequiring such treatment a therapeutically effective amount of a PAI-2variant of the first embodiment and/or a composition of the ninthembodiment.

According to a twelfth embodiment of this invention there is provided amethod of treatment of a fibrinolytic disorder, such as systemicfibrinolysis, comprising administering to a patient requiring suchtreatment a therapeutically effective amount of a PAI-2 variant of thefirst embodiment and/or a composition of the ninth embodiment.

According to a thirteenth embodiment of this invention there is provideda method of treatment of a condition such as multiple sclerosis, cornealor gastroduodenal ulceration, purpura, periodontitis, haemorrhage ormuscular dystrophy, comprising administering to a patient requiring suchtreatment a therapeutically effective amount of a PAI-2 variant of thefirst embodiment and/or a composition of the ninth embodiment.

According to a fourteenth embodiment of this invention there is provideda method for locating and/or defining the boundaries of a tumour in ahistological specimen or in vivo which method comprises applying aneffective amount of a labelled PAI-2 variant of the second embodiment tothe specimen or administering it to a host in need of in vivo imagingand determining by imaging, location of concentration of the label.

According to a fifteenth embodiment of this invention there is provideda method of improving the clinical efficacy of PA treatment ofthrombosis which method comprises administering a therapeuticallyeffective amount of a PAI-2 variant of the first embodiment and/or acomposition of the ninth embodiment to a host in need of such treatmentto counteract systemic activation of fibrinolysis and concomitantfibrin/fibrinogen breakdown.

According to a sixteenth embodiment of this invention there is providedan antibody against a PAI-2 variant of the first embodiment. Theantibody may be either a monoclonal or a polyclonal antibody.

The antibodies of the present invention can be used for detecting PAI-2and hence should be useful in the detection or monitoring of a number ofdisease states or conditions such as monocytic leukaemia, cancer, foetaldevelopment and chronic inflammatory diseases.

According to a seventeenth embodiment of this invention there isprovided a process for preparing an antibody of the sixteenthembodiment, which process comprises immunizing an immunocompetent hostwith an effective amount of a PAI-2 variant of the first embodimentand/or a composition of the ninth embodiment.

According to an eighteenth embodiment of this invention there isprovided an antibody composition comprising an antibody of the sixteenthembodiment together with a pharmaceutically acceptable carrier, diluentand/or excipient.

The antibody composition of the invention is of use in the detection ormonitoring of disease states or conditions for which the antibodies ofthe sixteenth embodiment can be used.

According to a nineteenth embodiment of this invention, there isprovided a diagnostic reagent comprising an antibody of the sixteenthembodiment and/or an antibody composition of the eighteenth embodiment.

According to a twentieth embodiment of this invention there is provideda conjugate comprising a variant of the first embodiment linked to acytotoxin. Examples of cytotoxins which may be used in the preparationof conjugates of the invention include: abrin; ricin; mellitin; gelonin;and the A sub unit from Diphtheria, tetanus, clostridial, Pertussis,Shigella, Pseudomonas, cholera or E. coli labile toxin.

Previous studies have demonstrated that human colon cancers producesignificantly greater amount of urokinase-type plasminogen activatorthan that occurring in adjacent non-involved tissue. PAI-2 has beenfound to be capable of binding to and inhibiting this tumour associatedplasminogen activator (Stephens et al. Blood 66 333-337, 1985). Thus, itfollows that biologically active PAI-2 variants have application asreagents for identifying and defining tumours both in vivo and inhistological specimens. For imaging tumours in vivo PAI-2 variants ofthe invention may be labelled with an appropriate isotope, such asTechnetium-99m (Richardson, V. J. Brit. J. Cancer 40; 35, 1979) orIodine-131 (Begent, R. H. J. Lancet, Oct. 2, 1982). Followingadministration of the PAI-2 variant preparation, the location andboundaries of the tumour may be determined by known radioisotopicmethods, such as gamma-camera imaging. Thus, PAI-2 variants offer asensitive method for enabling the identification of small metasticcancers particularly those arising after surgical intervention. In theanalysis of histochemical specimens, PAI-2 variants or antibodies raisedthereto, may be labelled with an isotope such as I¹³¹ or conjugated toan appropriate enzyme or other chemical reagent. On contact with ahistological specimen, such as a biopsy section, a PAI-2 variant of theinvention will bind to the tumour type plasminogen activator at itsplace of secretion, thereby identifying the tumour boundaries andpotentially the metastatic state of the tumour. In addition todiagnostic applications, PAI-2 variants are also indicated for use inthe direct treatment of tumours. As specific inhibitors of the enzymeimplicated in the process by which tumors invade surrounding tissues(Dano, K. et al., Adv. in Cancer Res. 44, 139, 1985), regulation and inparticular, inhibition of tumour growth and metastases can be achieved.Furthermore, PAI-2 variants can be used as a drug delivery system todeliver lectins or toxins directly to growing tumours. It will beappreciated that this system could offer many advantages in terms ofspecificity and extremely potent tumouricidal capability.

Other biological processes in which urokinase-type plasminogenactivators have been implicated involve those physiological eventsassociated with invasion and tissue destruction, such as chronicinflammatory conditions including rheumatoid arthritis. PAI-2 variantsare indicated to have a therapeutic effect when administered in vivo inameliorating such conditions.

According to a twenty-first embodiment of this invention there isprovided a cytotoxic composition comprising a conjugate of the twentiethembodiment together with a pharmaceutically acceptable carrier, diluentand/or excipient.

According to a twenty-second embodiment of this invention, there isprovided a method of delivering a cytotoxic agent to a tumour whichmethod comprises administering an effective amount of a conjugate of thetwentieth embodiment, and/or a cytotoxic composition of the twenty-firstembodiment to a host in need of such treatment.

According to a twenty-third embodiment of this invention there isprovided a diagnostic kit comprising a variant of the first embodimentand/or a composition of the ninth embodiment as a standard and anantibody of the sixteenth embodiment, an antibody composition of theeighteenth embodiment and/or a diagnostic reagent of the nineteenthembodiment.

The diagnostic kits of the invention are of use in the detection ormonitoring of diseases and conditions for which the antibodies of theinvention can be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 PAI-2 cDNA and amino acid sequence (SEQ ID NO: 1). The arrowsindicate the specific cleavage points so far identified.

FIG. 2 Bacterial (pBTA641) and baculovirus transfer vector (pAc373) usedin expression of PAI-2 and its variants in E. coli K-12 and insect cellsrespectively.

FIG. 3 Schematic representation of PAI-2 specific bands detected in abinding experiment with U-PA, two chain t-PA and single chain t-PA.

FIG. 4 Specific nucleotide and amino acid changes within PAI-2 to createthe deletion variants Δ66-98 (SEQ ID NO: 5) and Δ74-96 (SEQ ID NO: 3).The altered regions are described in: bases 214-222 of SEQ ID NO: 5 andamino acid residues 65-67 of SEQ ID NO: 6 for 66-98; bases 214-252 ofSEQ ID NO: 3 and amino acid residues 65-77 of SEQ ID NO: 4 for 74-96;and compared with the native sequence in bases 241-348 of SEQ ID NO: 1and amino acid residues 65-100 of SEQ ID NO: 2.

FIG. 5 Sequence of oligonucleotides A1 (SEQ ID NO: 7)and A2 (SEQ ID NO:10), used to create the deletion variant Δ74-96 in pBTA829.

FIG. 6 Construction of plasmid pBTA829 containing the deletion variant,Δ74-96. (Abbreviations used: B, BglII; H, HinfI; P, PstI; E, EcoRI;P_(L), leftwards promoter of bacteriophage lambda; T₇, promoter frombacteriophage T₇ ; Ap, ampicillin resistance gene; CIAP, calf intestinealkaline phosphatase)

FIG. 7 Schematic representation of PAI-2 specific bands (FIG. 7A) andurokinase specific bands (FIG. 7B) detected in a u-PA bindingexperiment.

FIG. 8 Sequence of oligonucleotides A134/301 (SEQ ID NO: 11), A134/304(SEQ ID NO: 12) and A134/305 (SEQ ID NO: 11), used to create thedeletion variant Δ66-98 in pBTA840. The location of the oligonucleotideswithin the PAI-2 coding region is indicated by the accompanying numbers.The numbering of the bases is as in FIG. 1.

Oligos A134/301 and A134/305 were used in a PCR reaction to generate aDNA fragment spanning the PAI-2 coding region from bases 235 to 541,with bases 244 to 342 inclusive deleted. Oligos A134/304 and the Sp6sequencing primer were used in a PCR reaction to generate a DNA fragmentspanning the PAI-2 coding region from bases 49 to 351, with bases 244 to342 inclusive deleted. Oligos A134/301 and the Sp6 sequencing primerwere used in a PCR reaction containing the products of the above two PCRreactions to generate a DNA fragment spanning the PAI-2 coding regionfrom bases 49 to 541, with bases 244 to 342 inclusive deleted.

FIG. 9 Construction of plasmid pBTA840 containing the deletion variant,Δ66-98. (Abbreviations used: B, Bgl II; E, EcoRI; P, Pst I; P_(L),leftwards promoter of bacteriophage lambda; Sp6, promoter frombacteriophage Sp6; T₇, promoter from bacteriophage T₇ ; Ap, ampicillinresistance gene; CIAP, calf intestine alkaline phosphatase; PCR,polymerase chain reaction).

FIG. 10 SDS-PAGE analysis of purified PAI-2 A66-98. 12% polyacrylamidegel (a.) and western (b.) (Anti-PAI-2 polyclonal antibodies) of thePAI-2 deletion mutant 66-98.

BEST MODE FOR CARRYING OUT THE INVENTION

The recombinant DNA molecules and transformed hosts of the invention areprepared using standard techniques of molecular biology.

Variants of the invention are obtained by culturing the transformedhosts of the invention under standard conditions as appropriate to theparticular host and separating the variant from the culture by standardtechniques. The variants may be used in impure form or may be purified.

Changes to PAI-2 can be made by modifying individual amino acids ofPAI-2 by site-directed mutagenesis of the DNA or by wholesalerestructuring by DNA deletion or insertion. These changes can beaccomplished in a variety of ways well known to those skilled in the art[e.g. "Molecular Cloning, A Laboratory Manual" Chapter 15 "Site-directedMutagenesis of cloned DNA" J. Sambrook, E. F. Fritsch, T. Maniatis (eds)1989; "Current Protocols in Molecular Biology" Chapter 8 "Mutagenesis ofCloned DNA" Ausubel, Brent, Kingston, Moore, Seidman, Smith and Struhl(eds) 1989], and include the use of oligonucleotides for point insertionand deletion mutagenesis, degenerate oligonucleotides for nestedmutations, the combing of long oligonucleotides to create a gene or genesegment with any desired changes, the use of Bal 31, DNAase I orExonuclease III to create deletion mutants, the use of chemicals and theuse of polymerase chain reaction (PCR). These techniques can be used toalter the 66-98 amino acid residue region of the PAI-2 molecule toproduce PAI-2 variants. The PAI-2 variants produced can then be screenedby the techniques described in Examples 1 and 2 to determine whetherparticular variants lack the protease sensitivity of PAI-2 in the 66-98amino acid residue region as evidenced by the absence of the 37 kD formand tested for maintenance of biological activity of PAI-2 as describedin Examples 1 and 2 for Δ66-98 and Δ74-96.

The compositions of the invention are prepared by mixing, preferablyhomogeneously mixing, variant with a pharmaceutically acceptablecarrier, diluent, and/or excipient using standard methods ofpharmaceutical preparation.

The amount of variant required to produce a single dosage form will varydepending upon the condition to be treated, host to be treated and theparticular mode of administration. The specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the variant employed, the age, body weight, general health,sex, and diet of the patient, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe condition undergoing treatment. The amounts required may bedetermined in accordance with standard pharmaceutical techniques.

The composition may be administered parenterally in unit dosageformulations containing conventional, non-toxic, pharmaceuticallyacceptable carriers, diluents and/or excipients as desired.

Injectable preparations of the variants of the invention, for example,sterile injectable aqueous or oleaginous suspensions may be formulatedaccording to the known arts using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

It is anticipated that it may be possible to deliver the variants of theinvention orally or topically as appropriate delivery systems aredeveloped.

Antibodies are raised using standard vaccination regimes in appropriatehosts. The host is vaccinated with a variant or composition of theinvention. The compositions used for vaccination purposes may include anadjuvant.

Suitable adjuvants for the vaccination of animals include but are notlimited to oil emulsions such as Marcol 52: Montanide 888 (Marcol is aTrademark of Esso. Montanide is a Trademark of SEPPIC, Paris), squalaneor squalene, Adjuvant 65 (containing peanut oil, mannide monooleate andaluminum monostearate), mineral gels such as aluminum hydroxide,aluminum phosphate, calcium phosphate and alum, surfactants such ashexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)propanediamine, methoxyhexadecylglycerol andpluronic polyols, polyanions such as pyran, dextran sulfate, polyacrylicacid and carbopol, peptides and amino acids such as muramyl dipeptide,dimethylglycine, tuftsin and trehalose dimycolate. The variants of thepresent invention can also be administered following incorporation intoliposomes or other micro-carriers, or after conjugation topolysaccharides, proteins or polymers. Other adjuvants suitable for usein the present invention include conjugates comprising the varianttogether with an integral membrane protein of prokaryotic or eukaryoticorigin, such as TraT.

Routes of administration, dosages to be administered as well asfrequency of injections are all factors which can be optimized usingordinary skill in-the art. Typically, the initial vaccination isfollowed some weeks later by one or more "booster" vaccinations, the neteffect of which is the production of high titres of antibodies againstthe variant.

Monoclonal antibodies against the variants of the invention can beprepared using standard techniques for monoclonal antibody production.

The antibody composition is prepared by mixing, preferably homogeneouslymixing, antibody with a pharmaceutically acceptable carrier, diluentand/or excipient using standard methods of pharmaceutical preparation.

Conjugates are prepared using standard techniques for conjugatesynthesis. The conjugate may be prepared chemically using linking agentsas necessary or by recombinant DNA techniques to provide a PAI-2 variantof the invention linked to a cytotoxic drug.

The conjugate composition is prepared by mixing, preferablyhomogeneously mixing, conjugate with a pharmaceutically acceptablecarrier, diluent and/or excipient using standard methods ofpharmaceutical preparation.

The amount of conjugate required to produce a single dosage form willvary depending upon the condition to be treated, host to be treated andthe particular mode of administration. The specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the conjugate employed, the age, body weight, generalhealth, sex, and diet of the patient, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe condition undergoing treatment. The amounts to be used can bedetermined by standard pharmaceutical techniques.

The conjugate composition may be administered parenterally, in unitdosage formulations containing conventional, non-toxic, pharmaceuticallyacceptable carriers, diluents, and/or excipients as desired.

Diagnostic kits are prepared by formulating antibodies at appropriateconcentration with a pharmaceutically acceptable carrier, diluent,and/or excipient. A positive control standard of a known concentrationof a variant of the invention is prepared similarly. The negativestandard comprises carrier, diluent, and/or excipient alone. Examples ofdiagnostic kits include a tumour diagnostic wherein the reagentcomprises an antibody of the invention and the positive controlcomprises a variant of the invention.

PLASMIDS

Various plasmids used in this work were derived from pBTA 438. PlasmidpBTA 438 consists of a 1.6kb cDNA encoding PAI-2 cloned in pUC18[Yanisch-Perron et al, Gene 33: 103-119 (1985)]. pBTA 438 was used totransform E. coli strain JM109 [Yanisch-Perron et al, Gene 33:103-119(1985)] to yield strain BTA 1445, which was deposited with the AmericanType Culture Collection of 12301 Parklawn Drive, Rockville, Md. 20852,USA under accession number ATCC 53585 on Feb. 11, 1987.

Plasmid pBTA641 can be derived from pBTA438 as follows. pBTA438 ispartially digested with XhoII plus DraI and a 1550bp fragment isolatedand ligated to vector pLK58 cut with BglII and SmaI. The resultantplasmid pBTA 446 was linearized with BglII and ligated to a syntheticdouble stranded 27 mer oligonucleotide having the sequence GATCT(N)₁₆ATGGAG (SEQ ID NO: 14), wherein N represents any nucleotide, containinga bacterial ribosome binding site and the initial nucleotides of thenative PAI-2 gene, creating plasmid pBTA641. Plasmid pBTA447 isidentical to pBTA 641 except that a 26 mer oligonucleotide containing abacterial ribosome binding site having the sequence GATCT(N)₁₅ ATGGAG(SEQ ID NO: 13) was used instead of the 27 mer.

Plasmid pMINS71 was derived as follows: the BglII-EcoRl PAI-2 genefragment from pBTA 641 was inserted into pSp72 (Promega) at theBglII/EcoRl sites; the BglII-SacI PAI-2 gene fragment from this vectorwas inserted into the HindIII/SacI sites of pGEM4Z (Promega) in a threeway ligation with a synthetic adaptor with cohesive HindIII-BglII endsto create pMINS71.

PREPARATIVE EXAMPLE 1

A. Bacterial Expression of PAI-2

Cell extracts of induced (by incubating cells at 42° C.) and uninduced(incubated at 30° C.) E. coli K-12 host cells containing pBTA447 andpBTA641 were screened for the presence of PAI-2 using affinity purifiedmonoclonal (Biopool) or polyclonal antibodies to human PAI-2. Biologicalactivity was assessed by a shift in the electrophoretic mobility in thepresence of urokinase, characteristic of the formation of aurokinase-PAI-2 complex. A PAI-2 protein band (M_(r) 46 kD), visualizedby western transfer using a monoclonal antibody to human placentalinhibitor and iodinated protein A, is present in the induced (42° C.)samples. A lower molecular weight (M_(r) 37,000) immunologicallycross-reactive protein band was also observed indicating possibleproteolytic cleavage of the PAI-2 molecule.

B. Purification of 37 kD Form

(i) Cell Growth and Lysis

E. coli K-12 cells harbouring the plasmid pBTA447 were heat induced at38° C. in a 10L fermenter for 24 h and the cells then recovered bycentrifuging at 17,000 xg for 20 min. A total of 524 g wet weight ofcells was recovered from 8L of fermentation broth.

The cells (524 g) were suspended in 1500 ml of 0.1M Na phosphate buffer,pH 7.0 containing 1 mM EDTA and 1 mM PMSF at 4° C. and lysed by fourpassages through a Martin-Gaulin press at 8000 psi. The press was washedout with 300 ml of the above buffer and the lysate and washes combined.To this solution was added MgCl₂ to a final concentration of 2 mM andthe solution centrifuged at 17,700 xg for 60 mins. The supernatant (1600ml) resulting from this centrifugation was recentrifuged at 30,100 xgfor 60 mins to remove remaining insoluble material and the supernatantrecovered. To this supernatant (1570 ml) was added 574.6 g of solidammonium sulphate to give a 60% saturated solution, the solution stirredfor 15 min. and then centrifuged at 30,000 xg for 30 mins. The resultantpellet, which contains the PAI-2 was divided into eighths and stored at-20° C.

(ii) DEAE-Sephacel Chromatography

One eighth aliquot of 0-60% ammonium sulphate precipitate was dissolvedin 200 ml of 0.1M Na phosphate, pH7.0 containing 1 mM EDTA and 0.1 MDTT, and incubated at 37° C. for 90 min. This solution was then dilutedto 500 ml with 0.1M Na phosphate, pH 7.0 containing 1 mM EDTA and 0.05%2-mercaptoethanol and dialysed at 4° C. against the same buffer for 48h. The dialysed solution was then applied to a DEAE-Sephacel column (4.4cm×10 cm) equilibrated in the above buffer and eluted until theabsorbance at 280 nm returned to base line. A linear 2 liter gradientfrom 0 to 0.5M NaCl in the same buffer was applied and the column elutedat a flow rate of 2 ml min⁻¹ Fractions of 10 ml were collected and 200μl aliquots analysed by SDS-PAGE and western analysis. The PAI-2 elutedbetween 58 mM and 81 mM NaCl under these conditions and these fractionswere pooled and dialysed against 1 mM Na phosphate, pH 7.0 containing0.05% 2-mercaptoethanol for 48 h at 4° C.

(iii) Hydroxylapatite Chromatography

The dialysed PAI-2 from the previous step was applied to a 3.2 cm×15 cmcolumn of Biogel HPT equilibrated in 1 mM Na phosphate, pH7.0 containing0.05% 2-mercaptoethanol and the column washed with the same buffer untilthe absorbance at 280 nm had returned to baseline. A one liter lineargradient from 1 mM Na phosphate to 200 mM Na phosphate was then appliedand the column eluted at a flow rate of 1 ml min⁻¹. Six ml fractionswere collected. The PAI-2 eluting from the column was detected usingSDS-PAGE and western blotting and revealed under reducing conditions twodistinct immunologically cross-reactive protein bands. The molecularweights of these two forms of PAI-2 were ca. 46 kD and ca. 37 kD.

(iv) High Pressure Liquid Chromatography

To resolve these two forms of PAI-2 an aliquot from the Biogel HPTcolumn containing PAI-2 was chromatographed on a Vydac C4 HPLC columnusing a gradient of acetonitrile in 0.1% TFA. This chromatographrevealed two major peaks, the former containing the 37 kD form of PAI-2and the latter containing the 46 kD form of PAI-2, as determined bynon-reducing SDS-PAGE. Amino acid sequencing of the Ca. 37 kD form ofPAI-2 revealed the sequence--Lys Gly Ser Tyr Pro Asp Ala Ile Leu Gln AlaGln Ala Ala Asp (SEQ ID NO: 16).

This sequence corresponds to a form of PAI-2 starting at residue 87 ofthe mature form of PAI-2 and suggests that the glutamine 86-lysine 87(Q86-K87) peptide bond is highly susceptible to proteolysis. A similarimmunologically cross reactive form of PAI-2 of ca. 37 kD was alsoobserved during the purification of naturally occurring PAI-2 from U937cells suggesting that proteolytic cleavage at the Q86-K87 peptide bondoccurs in both mammalian and bacterial cells and supporting the conceptthat this bond is highly labile.

C. Purification of 37 kD Form

E. coli cells harbouring the plasmid pBTA641 were heat induced at 38° C.in a 10 liter fermenter for 24 hours and the cells then recovered bycentrifugation at 17,000 xg for 20 mins.

(i) Cell Lysis

The cell pellet obtained from 5 liters of this fermentation wassuspended in 800 ml of 50 mM Na phosphate, containing 1 mM EDTA, 10 mMε-amino caproic acid (ε-ACA) and 10 mM 2-mercaptoethanol, pH 6.6, andlysed by six passages through a Martin-Gaulin 15 MR homogenizer at 9000psi. To the resultant lysate (900 ml) was added MgCl₂ to 2 mM and thesuspension centrifuged at 17,700 xg for 1 hour at 4° C.

(ii) Ammonium Sulphate Precipitation

To the supernatant from the above centrifugation was added solidammonium sulphate to give a 30% saturated solution. The solution wasstirred for 30 mins at 4° C. and then the precipitate removed bycentrifugation at 17,700 xg for 1 hour at 4° C. The supernatant (760 ml)was adjusted to 50% saturation by the addition of more solid ammoniumsulphate and following stirring at 4° C. for 30 min, the suspension wascentrifuged at 17,700 xg for 1 hour at 4° C. The pellet recovered fromthis precipitation step was dissolved in Buffer B (50 mM Na citrate, 1mM EDTA, 10 mM ε-ACA and 10 mM 2-mercaptoethanol, pH 5.5) to give afinal volume of 200 ml. This solution was then dialysed against 20volumes of Buffer B overnight at 4° C.

(iii) Phenyl Sepharose Chromatography

The dialysed solution was made 1M in ammonium sulphate and the sample(286 ml) was applied to a Phenyl Sepharose column (5 cm×19 cm; Vt=373ml), equilibrated in Buffer A, (Buffer A is Buffer B containing 1Mammonium sulphate) at a flow rate of 100 ml/h. Following loading of thesample the column was washed with Buffer A until the absorbance at 280nm (A280) returned to baseline and then a linear gradient of 800 ml ofBuffer A and 800 ml of Buffer B applied. Fractions of 10 ml werecollected. Following completion of the gradient the column was washedwith 50 mM glycine, pH9.0 until the A₂₈₀ returned to baseline. The PAI-2eluted in fraction 75-150 as determined by the urokinase inhibitionassay of Coleman and Green (in Methods in Enzymology 80: 408-414 1981)and by an immunological dot blot assay. These fractions were pooled (850ml) and precipitated by the addition of ammonium sulphate to 60%saturation. The pellet was recovered by centrifigation at 17,700 xg for30 mins at 4° C. and dissolved in Buffer C (25 mM Na borate, 1 mM EDTA,10 mM ε-ACA, 10 mM 2-mercaptoethanol, pH9.0).

(iv) Sephacryl S200 Chromatography

The solution containing PAI-2 (25 ml) was applied to a Sephacryl S200column (3.3 cm×95 cm) equilibrated in Buffer C and eluted at a flow rateof 40 ml/h. Fractions of 6 ml were collected and analysed for PAI-2 byurokinase inhibition, SDS-PAGE and immunological cross-reactivity in adot blot assay. Fractions containing the PAI-2 (fractions 52-90) werepooled and precipitated with 60% saturated ammonium sulphate. The pelletwas recovered by centrifugation at 17,700 xg for 30 mins at 4° C. andredissolved in Buffer A. The pH of this solution was adjusted to 5.5with HCl and the precipitate which developed was removed bycentrifugation at 17,700 xg for 30 mins at 4° C.

(v) Second Phenyl Sepharose Chromatography

The supernatant was applied to a Phenyl Sepharose column (5 cm×10 cm) ata flow rate of 60 ml/h. Following loading, the column was washed withBuffer A and then a linear gradient of Buffer A and Buffer B applied asdescribed in (iii) above. Following completion of the gradient thecolumn was washed with 50 mM glycine, pH9 and the fractions containingPAI-2 identified by SDS-PAGE and western blotting. Fractions 15-27,containing the PAI-2, were pooled, precipitated with 60% ammoniumsulphate and redissolved in 15 ml of Buffer C.

(vi) Second Sephacryl S 200 Chromatography

The sample containing PAI-2 from the second Phenyl Sepharose columnabove was applied to a Sephacryl S200 column (2.5 cm×95 cm) equilibratedin Buffer C and eluted at a flow rate of 30 ml/h. Fractions of 2.6 mlwere collected and analysed for PAI-2 by SDS-PAGE.

(viii) Reverse Phase HPLC

The SDS-PAGE of the fractions from the second Sephacryl S200 columnshowed the presence of two proteins with approximate molecular weightsof ca 46 kD and 37 kD when electrophoresed in the presence of2-mercaptoethanol. These protein bands are similar to those observed in"B. Purification of 37 kD Form". To resolve these two forms, a 90 μlaliquot of fraction 106 from the second Sephacryl S-200 column above waschromatographed on a Vydac C₄ reverse phase HPLC column using a gradientof acetonitrile in 0.1% TFA. The leading edge of the major absorbancepeak eluted from this column contained primarily the 37 kD protein.Amino acid sequencing of this fraction revealed an N-Terminal sequenceof F M Q Q I Q K G S Y (Phe Met Gln Gln Ile Gln Lys Gly Ser Tyr: SEQ IDNO: 17) which corresponds to the sequence of PAI-2 starting at aminoacid residue 81. It was therefore concluded that this form of PAI-2arose from proteolytic cleavage of the mature form of PAI-2 at theglycine 80-phenylalanine 81 bond.

The observation that purification of PAI-2 overexpressed in E. coli bythis alternative method and, in particular, the inclusion of ε-ACA as aninhibitor of lysine specific proteases, protected PAI-2 from cleavage atthe Q86-K87 bond but not cleavage at a region only six amino acidsupstream of this site, reinforces the view that this region of themolecule is highly susceptible to protease cleavage.

D. Purification of 37 kD Form

To determine whether the proteolysis observed above could be preventedby expression in an alternative host PAI-2 was overexpressed in thebaculovirus insect cell system (Lucknow and Summers, Biotechnology 6:47-55, 1988). The expressed product was purified essentially asdescribed in "C. Purification of 37 kD Form" using steps (i) through(iv) except that the 50% ammonium sulphate precipitation step in (ii)was omitted. The PAI-2 eluting from the Sephacryl S-200 column wasdetected by SDS-PAGE and western blotting under reducing conditions.This analysis showed the presence of both a 46 kD and a 37 kD form ofPAI-2, indicating that proteolytic cleavage of the molecule wasoccurring as observed previously. To further define the site of thiscleavage the PAI-2 pool obtained from the Sephacryl S-200 chromatographystep was dialysed against 20 mM glycine, 10 mM EDTA and 10 mM2-mercaptoethanol, pH9.0 and the sample (30 ml) then applied to aQ-Sepharose column (0.9 cm×24 cm) at a flow rate of 60 ml/h. The PAI-2eluted unretarded from this column and on SDS-PAGE revealed twoCoomassie blue staining bands of Mr=ca 37 kD and ca 46 kD. N-terminalamino acid sequencing of an aliquot of this material revealed a singlesequence as shown below G F M Q Q I Q K G S Y P D A I (i.e. Gly Phe MetGln Gln Ile Gln Lys Gly Ser Tyr Pro Asp Ala Ile: SEQ ID NO: 18). Noauthentic N-terminal sequence for the full length PAI-2 was observed,indicating that the 46 kD form of PAI-2 when expressed in insect cellscontains a blocked N-terminus. Similar results have been observed withfull length PAI-2 isolated from U937 cells (Kruithof et al J. Biol Chem216: 11207-11213 1986) and from placenta (Andreasen et al 261: 7644-76511986). The observed sequence is consistent with proteolytic cleavageoccurring between cysteine 79 and glycine 80, only one peptide bondupstream from the G80-F81 cleavage site observed with PAI-2 purifiedfrom E. coli in "C. Purification of 37 kD Form".

These results further confirm the high degree of proteolyticsusceptibility of this region of the PAI-2 molecule. E. Purification ofthe K87A variant of PAI-2

Creation of a variant PAI-2, wherein amino acid residue 87 was changedfrom Lys to Ala, was achieved by site-directed mutagenesis, aftertransferring the PAI-2 coding region to the phage M13 vector mp18.Preparation and use of single-stranded phage DNA, as well as the use ofthe two oligonucleotides containing the mutated sequence [(5'-CAG CAGATC CAG GCA GGT AGT TAT CCT-3' (SEQ ID NO: 19), 5'-AGG ATA ACT ACC TGCCTG GAT CTG CTG-3' (SEQ ID NO: 21) complement of SEQ ID NO: 19; with theamino acid translation shown in (SEQ ID NO: 20) ], were carried out aspreviously described (Amersham; oligonucleotide-directed in vitromutagenesis system).

The K87A variant of PAI-2 was purified from a 1 liter culture of E. coliK-12 cells harbouring the plasmid pBTA674. This plasmid is identical topBTA641 but with the PAI-2 DNA replaced with the variant form of PAI-2.

The purification was performed essentially as described in "C.Purification of 37 kD Form", steps (i) through (iv) except that the 50%ammonium sulphate precipitation in step (ii) was omitted. Analysis ofthe fractions eluted from the Sephacryl S-200 column by reducingSDS-PAGE and western blotting indicated the presence of both a 46 kD anda 37 kD form of PAI-2, indicating that mutagenesis of lysine 87 to analanine residue failed to prevent cleavage of the PAI-2 molecule in theregion previously identified as protease sensitive (see B. Purificationof 37 kD form).

EXAMPLE 1

Deletion of Protease Sensitive Site

The HinfI-PstI segment spanning the protease sensitive site in PAI-2 wasreplaced by synthetic oligonucleotides with cohesive HinfI and PstI ends(see FIG. 5), creating a variant PAI-2 in which amino acids 74 to 96inclusive were deleted. The events involved in the construction of thisdeletion variant are illustrated in FIG. 6. In essence, the deletionvariant was assembled in an intermediate vector by a three way ligationbetween a BglII-HinfI fragment from pBTA641, a PstI-BglII fragment frompMINS71 and the annealed oligonucleotides A1 (SEQ ID NO: 7) and A2 (SEQID NO: 8). The assembled deletion PAI-2 was then excised from theintermediate vector and exchanged with native PAI-2 in pBTA641 to createpBTA 829.

Oligonucleotides (FIG. 5 SEQ ID NOS: 7 and 8) were synthesized on anApplied Biosystems DNA synthesizer (Model 380A), and purified through apolyacrylamide gel. Complementary oligonucleotides (A1: SEQ ID NO: 7 andA2 SEQ ID NO: 8) were mixed in a 1:1 molar ratio and phosphorylatedusing 5 units T4 polynucleotide kinase in 65 mM Tris-Cl pH 7.5, 10 mMMgCl₂, 5 mM dithiothreitol, 1 mM ATP. The mixture was heated to 65° C.for 10 minutes and cooled slowly to room temperature to allow annealingto take place. The various restriction fragments were prepared asfollows. Restriction enzyme digests of purified plasmid DNA were carriedout in buffers recommended by the supplier. Required DNA fragments wereseparated from the plasmid by gel electrophoresis through 0.8-1.5%Sea-Plaque agarose (FMC Corporation) in Tris-acetate buffer (Maniatis etal, 1982). Fragments were visualized by staining with ethidium bromideand UV transillumination. The band of agarose containing the appropriatefragment was sliced out of the gel, melted at 65° C. and the DNA wasextracted three times with phenol/chloroform/isoamyl alcohol. The DNAwas then precipitated with ethanol.

Vectors were typically prepared as follows. Plasmid DNA was digestedwith the appropriate restriction enzymes, the digest was extracted withan equal volume of phenol/chloroform/isoamyl alcohol and the DNAprecipitated with 2.5 volumes of ethanol. The digested DNA wasresuspended in 50 mM Tris-Cl pH 9.0, 1 mM MgCl₂, 0.1 mM ZnCl₂, 1 mMspermidine and incubated with 1-2 units calf intestinal alkalinephosphatase (Boehringer Mannhelm) for 30-60 mins at 37° C. The enzymewas heat killed at 70° C. for 15 minutes then the DNA was extracted withphenol/chloroform/isoamyl alcohol and precipitated with ethanol.

Ligations were carried out as follows. Vector and insert DNAs were mixedat a molar ratio of between 1:1 and 1:5 (1:10 if the insert was smallerthen 100 bp) in 1 mM ATP, 10 mM MgCl₂, 5 mM DTT, 65 mM Tris-Cl-pH7.5 ina volume of 20 [l. Ligations were carried out at 16° C. overnight with0.5-1 unit T4 DNA ligase (Boehringer Mannhelm). From ligation mixes 5-10μl was removed for transformation into a competent E. coli K12 host(Hanahan, J. Mol Biol 166: 557-580, 1983). Transformants were selectedby plating onto tryptone-soya agar plates containing 100 μg/mlampicillin.

Plasmid DNA was extracted from individual colonies and the correctrecombinant plasmids identified by restriction analysis. The region ofthe PAI-2 gene where the deletion was made was sequenced to confirm thechanges. Sequencing was carried out on double-stranded plasmid DNA usingthe Sequenase DNA Sequencing Kit (USB) as described in the instructionmanual. The primer used was the T7 primer (Promega).

Bacterial Constructions and Expression

The complete coding sequence of PAI-2 and the deletion variant wereplaced under the control of the lambda P_(L) promoter in the vectorpLK58, with a synthetic oligonucleotide upstream of the ATG providing abacterial ribosome binding site at an appropriate distance from thestart codon, giving plasmids pBTA 641 encoding the native PAI-2 sequenceand pBTA829 encoding the deletion variant Δ74-96.

These plasmids were used to transform an E. coli K-12 ΔHI Δtrp hostwhich contained the thermolabile repressor of lambda, cI857. Transformedcells were grown overnight in TSB medium (Oxoid) at 28° C. Cells werethen diluted in MEB medium (Mott et al Proc. Natl. Acad. Sci. 82: 88-921985), grown at 28° C. to an OD₆₀₀ of 1.0 when prewarmed (48° C.) MEBmedium was added in equal volume to equilibrate the temperature to 38°C. Following 4 hours of growth at 38° C. the cells were harvested bycentrifugation at 8000×g for 15 mins. Cell pellets were resuspended inBehs buffer (10 mM p-chloromercuro benzoic acid, 10 mM EDTA (Na)₂, 10 mM1,10-phenathioline, 100 mM phosphate, pH7.0) and lysed by two passesthrough a french press at 16,000 psi (on ice). The supernatant fromlysed cells was clarified by centrifugation at 8000 xg for 15 mins andtested for the presence of PAI-2 using affinity purified monoclonal(Biopool) or polyclonal antibodies to human PAI-2. Biological activitywas also assessed by a shift in the electrophoretic mobility in thepresence of urokinase, characteristic of the formation of aurokinase--PAI-2 complex, as described below.

U-PA Binding Experiment

The ability of the deletion variant of PAI-2 described above to bind tourokinase was determined in a urokinase binding experiment. Since theΔ74-96 variant is a significantly altered molecule compared to thenative PAI-2 it is not possible to predict whether the variant hasbiological activity or not. Urokinase (LMW, American Diagnostica) wasadded to clarified supernatant from lysed cells expressing native (i.e.expressed from pBTA641) or variant Δ74-96 (i.e. expressed from pBTA829)PAI-2, or no PAI-2 (i.e. cells containing pBTA 836). As a negativecontrol, lysates were used without the addition of urokinase. PlasmidpBTA 836 was derived from pBTA 641 by digestion with BglII and EcoRl toexcise the PAI-2 gene, followed by a fill-in reaction using Klenowenzyme and a ligation reaction to reform an intact plasmid lacking thePAI-2 gene.

Binding was allowed to proceed at room temperature for 90 minutes.Samples were then boiled for 3 minutes after the addition of a buffercontaining SDS and 2-mercaptoethanol and analysed by SDS-PAGE andwestern blotting, using either a goat polyclonal antibody against PAI-2(FIG. 7A), or rabbit polyclonal antibody against urokinase (FIG. 7B).Bound antibody was detected using a second antibody-HRP conjugatedirected against the primary antibody.

FIG. 7 shows the results of such an experiment. The addition ofurokinase to lysates containing native or variant Δ74-96 PAI-2 resultedin the formation of an SDS stable complex of approximately 69 kD thatreacted with either polyclonal antibody directed against PAI-2 (FIG. 7A,lanes c and e) or antibody directed against urokinase (FIG. 7B, lanes cand e). In the absence of urokinase, or in cell lysates lacking PAI-2,such a complex could not be detected using either antibody against PAI-2(FIG. 7A, lanes b, d, f, g) or antibody against urokinase (FIG. 7B,lanes b, d, f, g). These results are characteristic of the formation ofa urokinase-PAI-2 complex and indicate that both the native PAI-2 andthe variant Δ74-96 PAI-2 are capable of binding urokinase and hencepossess biochemical activity.

Elimination of Proteolytic Sensitivity

In E. coli cells expressing native PAI-2 (i.e. from pBTA 641) the majorproducts detected by PAI-2 specific antibody following SDS-PAGE andwestern transfer are a 46 kD form, representing native PAI-2, and a 37kD form representing a degradation product (FIG. 7A, lane b). In cellsexpressing the variant A74-96 PAI-2 (i.e. from pBTA 829) the 37 kDdegradation product cannot be detected (FIG. 7A, lane d). These resultsshow that the variant Δ74-96 PAI-2 does not possess the proteolyticsensitivity of the native PAI-2.

EXAMPLE 2

Deletion of Protease Sensitive Site

DNA sequences encoding amino acids 66-98 inclusive were deleted from thePAI-2 coding region using the polymerase chain reaction (PCR) techniqueof site-directed mutagenesis by overlap extension (Ho et al. Gene 77:51-59, 1989). The oligonucleotides used in the PCR reactions are shownin FIG. 8 (SEQ ID NOS: 9, 10 and 11) and an outline of the constructionof this deletion variant illustrated in FIG. 9.

In brief, the PAI-2 DNA was transferred to an intermediate vector whichwas used in PCR reactions to generate a Bgl II/PstI fragment in whichthe sequences encoding amino acids 66-98 inclusive had been deleted. ThePCR generated Bgl II/Pst I deletion fragment was exchanged for thenative Bgl II/Pst I fragment in the intermediate vector and the entireBgl II/Pst I region from five independent transformants sequenced. Fromone of these transformants, in which the only differences from thenative PAI-2 was the deletion of sequences encoding amino acids 66-98inclusive, the PAI-2 DNA was recovered and ligated into the vector pLK58 to create pBTA 840.

Oligonucleotides (FIG. 8: SEQ ID NOS: 9, 10 and 11) were synthesized onan Applied Biosystems DNA synthesizer (Model 380A), with the tritylgroup left on, and purified on oligonucleotide purification cartridges(Applied Biosystems, Cat. No. 400771) according to the manufacturer'sinstructions. One oligo, Sp6 primer, was purchased from Promega.

PCR reactions were in 50 mM KCl, 10 mM tris-HCl pH 8.3, 1.5 mM Mg Cl₂,0.01% gelatin w/v, 200 μM dNTPs, 2.5U amplitaq (Perkin-Elmer Cetus),using 100 pmoles of oligonucleotides and 0.35 pmoles of Eco R1linearized PAI-2 plasmid. Reactions were carried out on a Gene Machine(Innovonics) set for 25 cycles with 1 minute denaturation (94° C.), 1minute annealing (50° C.) and 1 minute extension (74° C.). PCR productswere separated from oligonucleotides either on Sephacryl S-200 columnsor by gel electrophoresis through 1.5% sea-plaque agarose (FMCCorporation) in tris-acetate buffer (Maniatis et al 1982), followed bystaining with ethidium bromide, visualization on a UV transilluminatorand purification from the agarose gel slice on NACS columns (BRL)according to the manufacturer's instructions.

Other required DNA fragments were separated from plasmid DNA by gelelectrophoresis through 0.8-1.5%, sea-plaque agarose and purified asdescribed above for PCR products.

Vectors were typically prepared as follows. Plasmid DNA was digestedwith the appropriate restriction enzymes for 1 to 2 hrs. at 37° C. Calfintestinal alkaline phosphatase (CIAP Boehringer Mannhelm, 1 to 2 units)was added directly to the restriction digest and the incubationcontinued at 37° C. for 1 hour. In some cases, when restriction enzymesthat yielded flush or 3' overhang ends were used, the incubation withCIAP was for 30 minutes at 37° C. and 30 minutes at 50° C. The CIAPenzyme was heat killed at 70° C. for 15 minutes and the DNA wasextracted with phenol/chloroform/isoamyl alcohol and precipitated withethanol.

Ligations and transformation of E. coli K12 hosts were as described inExample 1. In some cases ligations were at 4° C. for 48 hours or 16° C.for 4 to 6 hours.

Sequencing of the Bgl II/Pst I regions were performed on double-strandedplasmid DNA, after alkali denaturation, using a Multiwell MicrotitreSequencing System Kit (Amersham) as described in the instruction manual.The primer used was the Sp6 primer (Promega).

Plasmids used in this-work were derived as described above.

Expression in E. coli

Plasmid pBTA 840 was used to transform an E. coli ΔH₁ Δtrp host whichcontained the thermolabile repressor of lambda, cI857. A singletransformant was grown overnight in TSB medium at 28° C. and theresulting culture used to innoculate a 10 liter fermenter. The E. colicells were heat induced at 38° C. for 24 hrs. and the cells recovered bycentrifugation at 17,000 g for 20 min.

Purification of Δ66-98 and Δ74-96 PAI-2

The PAI-2 variants Δ66-98 and Δ74-96 can be purified from cells of E.coli expressing the molecule using a combination of the procedures usedin the purification of the native molecule viz processes involvingphenyl-sepharose chromatography, Sephacryl S200 chromatography, ionexchange chromatography and/or reverse phase HPLC. These procedures aredescribed in International Patent Application No PCT/AU85/00191 (WO86/01212) and International Patent Application No PCT/AU87/00068 (WO87/05628) and the results of purifying Δ66-98 are illustrated in FIG.10.

U-PA, two chain t-PA, and single chain t-PA binding experiment

The ability of the purified deletion variant Δ66-98 to bind to U-PA, totwo chain t-PA and to single chain t-PA was examined in a bindingexperiment similar to that described in Example 1. The bindingcharacteristics of Δ66-98 PAI-2 (SEQ ID NO: 3) were compared to thoseexhibited by native PAI-2 (i.e. as expressed from pBTA641) (SEQ ID NO:1), the Δ74-96 variant (i.e. as expressed from pBTA829) (SEQ ID NO: 2)and to the second form of native PAI-2 that differs by three amino acidsfrom the PAI-2 expressed from pBTA641 (Schleuning et. al. Mol. Cell.Biol. 2: 4564-4567, 1987). The alternative native PAI-2 was expressedfrom pBTA 683. Plasmid pBTA 683 was derived from pBTA641 by sitedirected mutagenesis that changed 3 amino acids to that found in thesecond form of PAI-2. [Schleuning et al. Mol. Cell Biol. 7: 4564-4567(1987 )].

The various PAI-2s (0.25 μg each) were incubated with either u-PA (3.75μg, Behring), two chain t-PA or single chain t-PA (3.75 μg each,American Diagnostica) at room temperature for 160 minutes in 25 mMTris-HCl pH7.5, 75 mM NaCl, 2.5 mM EDTA and 0.5% TX-100. Samples wereanalysed on 10% SDS-polyacrylamide gels followed by western blotting.Blots were probed with a goat polyclonal antibody against PAI-2 andbound antibody detected by an anti-goat-HRP conjugate (FIG. 11).

All PAI-2s, (the two native forms and the two deletion variants)displayed identical binding characteristics. Thus, on incubation witheither U-PA, two chain t-PA or single chain t-PA high molecular weightSDS stable forms of PAI-2 were seen. Such high molecular weight formsare characteristic of the formation of complexes between PAI-2 and theseplasminogen activators.

Elimination of Proteolytic Sensitivity

As with the variant Δ74-96, the 37 kD degradation product observed onpurification of native PAI-2, was not found in purified preparations ofthe variant Δ66-98 (FIG. 10).

INDUSTRIAL APPLICABILITY

The PAI-2 variants of the invention can be used as therapeutic anddiagnostic agents in patients with tumours, or suffering from chronicinflammatory conditions such as rheumatoid arthritis.

Other conditions where the application of a specific PA inhibitor may beof use include diseases or conditions such as osteoarthritis, multiplesclerosis, colitis ulcerosa, SLE-like disease, psoriasis, pemphigus,corneal ulcer, gastroduodenal ulcer, purpura, periodontitis, haemorrhageand muscular dystrophy. A specific PA inhibitor would also be useful asan adjunct to thrombolytic therapy involving PAs in order to reduce theincidence and severity of the side effect of such treatment viz.systemic fibrinolysis. Finally, a PA inhibitor could have a significantrole in skin wound healing and tissue repair especially since twotrypsin inhibitors have been shown to enhance formation of connectivetissue with increased tensile strength of the wound tissue [Kwaan, HCand Astrup, T (1969) Exp. Molec. Path 11, 82] and keratinocytes areknown to produce both uPA and tPA [Grondahl-Hansen, J et al. (1988) J.Invest Dermatol.].

Antibodies against variants of the invention should be useful in thedetection or monitoring of disease states or conditions such asmonocytic leukaemia, cancer, foetal development and chronic inflammatorydiseases.

We claim:
 1. A plasminogen activator inhibitor type 2 variant in whichthe 66-98 amino acid residue region of SEQ ID NO: 2 has been altered toeliminate at least one protease sensitive site, which variant maintainsbiological activity of plasminogen activator inhibitor type 2 of SEQ IDNO: 2 amino acids up to 65 and from 99 of plasminogen activatorinhibitor type 2 in frame.
 2. A plasminogen activator inhibitor type 2variant according to claim 1, which variant is a deletion variant inwhich at least one amino acid residue in the 66-98 region of SEQ ID NO:2 has been deleted.
 3. The plasminogen activator inhibitor type 2variant according to claim 1, wherein said variant is variant Δ74-96 ofSEQ ID NO: 4 in which amino acids 74-96 inclusive of plasminogenactivator inhibitor type 2 have been deleted.
 4. The plasminogenactivator inhibitor type 2 variant according to claim 1, wherein saidvariant is variant Δ66-98 of SEQ ID NO: 6 in which amino acids 66-98inclusive of plasminogen activator inhibitor type 2 have been deleted.5. A plasminogen activator inhibitor type 2 variant according to claim1, wherein said variant is labelled with one of a radioisotope and achemical agent.
 6. A diagnostic composition comprising at least oneplasminogen activator inhibitor type 2 variant according to claim 1together with a pharmaceutically acceptable carrier, excipient, ordiluent.
 7. A diagnostic composition comprising at least one plasminogenactivator inhibitor type 2 variant according to claim 5 together with apharmaceutically acceptable carrier, excipient, or diluent.
 8. Anantibody specific to a plasminogen activator inhibitor type 2 variantaccording to claim
 1. 9. A polyclonal antibody according to claim
 8. 10.A monoclonal antibody according to claim
 8. 11. A process for preparingan antibody specific to a plasminogen. activator inhibitor type 2variant according to claim 1, which process comprises immunizing animmuno-competent host with an effective amount of a a plasminogenactivator inhibitor type 2 variant according to claim
 1. 12. An antibodycomposition comprising an antibody according to claim 8 together with apharmaceutically acceptable carrier, diluent, or excipient.
 13. Adiagnostic reagent comprising an antibody according to claim 8 or anantibody according to claim 8 together with a pharmaceuticallyacceptable carrier, diluent, or excipient.
 14. A diagnostic kitcomprising any of the following: a variant according to claim 1 as astandard, or an antibody specific to a plasminogen activator inhibitortype 2 variant according to claim
 1.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1610 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (G) CELL TYPE: Monocyte                                                       (H) CELL LINE: U937                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: BTA 1445                                                           (viii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT: 18                                                    (B) MAP POSITION: 18q21-q23                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 49..1296                                                         (D) OTHER INFORMATION: /product="human plasminogen                           activator inhibitor type 2 protein"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTCAGACAGCAACTCAGAGAATAACCAGAGAACAACCAGATTGAAACAATGGAGGAT57                   MetGluAsp                                                                      1                                                                            CTTTGTGTGGCAAACACACTCTTTGCCCTCAATTTATTCAAGCATCTG105                           LeuCysValAlaAsnThrLeuPheAlaLeuAsnLeuPheLysHisLeu                               51015                                                                        GCAAAAGCAAGCCCCACCCAGAACCTCTTCCTCTCCCCATGGAGCATC153                           AlaLysAlaSerProThrGlnAsnLeuPheLeuSerProTrpSerIle                              20 253035                                                                     TCGTCCACCATGGCCATGGTCTACATGGGCTCCAGGGGCAGCACCGAA201                           SerSerThrMetAlaMetValTyrMetGlySerArgGlySerThrGlu                               404550                                                                       GACCAGATGGCCAAGGTGCTTCAGTTTAATGAAGTGGGAGCCAATGCA249                           AspGlnMetAlaLysValLeuGlnPheAsnGluValGlyAlaAsnAla                               556065                                                                       GTTACCCCCATGACTCCAGAGAACTTTACCAGCTGTGGGTTCATGCAG297                           ValThrProMetThrProGluAsnPheThrSerCysGlyPheMetGln                              70 7580                                                                       CAGATCCAGAAGGGTAGTTATCCTGATGCGATTTTGCAGGCACAAGCT345                           GlnIleGlnLysGlySerTyrProAspAlaIleLeuGlnAlaGlnAla                              85 9095                                                                       GCAGATAAAATCCATTCATCCTTCCGCTCTCTCAGCTCTGCAATCAAT393                           AlaAspLysIleHisSerSerPheArgSerLeuSerSerAlaIleAsn                              100105 110115                                                                 GCATCCACAGGGAATTATTTACTGGAAAGTGTCAATAAGCTGTTTGGT441                           AlaSerThrGlyAsnTyrLeuLeuGluSerValAsnLysLeuPheGly                              120 125130                                                                    GAGAAGTCTGCGAGCTTCCGGGAAGAATATATTCGACTCTGTCAGAAA489                           GluLysSerAlaSerPheArgGluGluTyrIleArgLeuCysGlnLys                              135 140145                                                                    TATTACTCCTCAGAACCCCAGGCAGTAGACTTCCTAGAATGTGCAGAA537                           TyrTyrSerSerGluProGlnAlaValAspPheLeuGluCysAlaGlu                              150 155160                                                                    GAAGCTAGAAAAAAGATTAATTCCTGGGTCAAGACTCAAACCAAAGGC585                           GluAlaArgLysLysIleAsnSerTrpValLysThrGlnThrLysGly                              165170 175                                                                    AAAATCCCAAACTTGTTACCTGAAGGTTCTGTAGATGGGGATACCAGG633                           LysIleProAsnLeuLeuProGluGlySerValAspGlyAspThrArg                              180185190 195                                                                 ATGGTCCTGGTGAATGCTGTCTACTTCAAAGGAAAGTGGAAAACTCCA681                           MetValLeuValAsnAlaValTyrPheLysGlyLysTrpLysThrPro                              200205 210                                                                    TTTGAGAAGAAACTAAATGGCCTTTATCCTTTCCGTGTAAACTCGGCT729                           PheGluLysLysLeuAsnGlyLeuTyrProPheArgValAsnSerAla                              215220 225                                                                    CAGCGCACACCTGTACAGATGATGTACTTGCGTGAAAAGCTAAACATT777                           GlnArgThrProValGlnMetMetTyrLeuArgGluLysLeuAsnIle                              230235 240                                                                    GGATACATAGAAGACCTAAAGGCTCAGATTCTAGAACTCCCATATGCT825                           GlyTyrIleGluAspLeuLysAlaGlnIleLeuGluLeuProTyrAla                              245250255                                                                      GGAGATGTTAGCATGTTCTTGTTGCTTCCAGATGAAATTGCCGATGTG873                          GlyAspValSerMetPheLeuLeuLeuProAspGluIleAlaAspVal                              2602652702 75                                                                 TCCACTGGCTTGGAGCTGCTGGAAAGTGAAATAACCTATGACAAACTC921                           SerThrGlyLeuGluLeuLeuGluSerGluIleThrTyrAspLysLeu                              280285 290                                                                    AACAAGTGGACCAGCAAAGACAAAATGGCTGAAGATGAAGTTGAGGTA969                           AsnLysTrpThrSerLysAspLysMetAlaGluAspGluValGluVal                              295300305                                                                     TACATACCCCAGTTCAAATTAGAAGAGCATTATGAACTCAGATCCATT1017                          TyrIleProGlnPheLysLeuGluGluHisTyrGluLeuArgSerIle                              310315320                                                                     CTG AGAAGCATGGGCATGGAGGACGCCTTCAACAAGGGACGGGCCAAT1065                         LeuArgSerMetGlyMetGluAspAlaPheAsnLysGlyArgAlaAsn                              325330335                                                                     TTCTCAGGGATG TCGGAGAGGAATGACCTGTTTCTTTCTGAAGTGTTC1113                         PheSerGlyMetSerGluArgAsnAspLeuPheLeuSerGluValPhe                              340345350355                                                                  CACCAAGC CATGGTGGATGTGAATGAGGAGGGCACTGAAGCAGCCGCT1161                         HisGlnAlaMetValAspValAsnGluGluGlyThrGluAlaAlaAla                              360365370                                                                     GGCACAG GAGGTGTTATGACAGGGAGAACTGGACATGGAGGCCCACAG1209                         GlyThrGlyGlyValMetThrGlyArgThrGlyHisGlyGlyProGln                              375380385                                                                     TTTGTGGCA GATCATCCTTTTCTTTTTCTTATTATGCATAAGATAACC1257                         PheValAlaAspHisProPheLeuPheLeuIleMetHisLysIleThr                              390395400                                                                     AACTGCATTTTATTT TTCGGCAGATTTTCCTCACCCTAAAACTAAG1303                           AsnCysIleLeuPhePheGlyArgPheSerSerPro                                          405410415                                                                     CGTGCTGCTTCTGCAAAAGATTTTTGTAGATGAGCTGTGTGC CTCAGAATTGCTATTTCA1363             AATTGCCAAAAATTTAGAGATGTTTTCTACATATTTCTGCTCTTCTGAACAACTTCTGCT1423              ACCCACTAAATAAAAACACAGAAATAATTAGACAATTGTCTATTATAACATGACAACCCT1483              ATTAATCATTTGGTCTTCTA AAATGGGATCATGCCCATTTAGATTTTCCTTACTATCAGT1543             TTATTTTTATAACATTAACTTTTACTTTGTTATTTATTATTTTATATAATGGTGAGTTTT1603              TAAATTA161 0                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 415 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGluAspLeuCysValAlaAsnThrLeuPheAlaLeuAsnLeuPhe                              15 1015                                                                       LysHisLeuAlaLysAlaSerProThrGlnAsnLeuPheLeuSerPro                              202530                                                                        TrpSerIleSerSerThrMetAlaMetV alTyrMetGlySerArgGly                             354045                                                                        SerThrGluAspGlnMetAlaLysValLeuGlnPheAsnGluValGly                              505560                                                                        Ala AsnAlaValThrProMetThrProGluAsnPheThrSerCysGly                             65707580                                                                      PheMetGlnGlnIleGlnLysGlySerTyrProAspAlaIleLeuGln                               859095                                                                       AlaGlnAlaAlaAspLysIleHisSerSerPheArgSerLeuSerSer                              100105110                                                                     AlaIleAsnAlaSerTh rGlyAsnTyrLeuLeuGluSerValAsnLys                             115120125                                                                     LeuPheGlyGluLysSerAlaSerPheArgGluGluTyrIleArgLeu                              130135 140                                                                    CysGlnLysTyrTyrSerSerGluProGlnAlaValAspPheLeuGlu                              145150155160                                                                  CysAlaGluGluAlaArgLysLysIleAsnSerTrpValLysT hrGln                             165170175                                                                     ThrLysGlyLysIleProAsnLeuLeuProGluGlySerValAspGly                              180185190                                                                     AspThr ArgMetValLeuValAsnAlaValTyrPheLysGlyLysTrp                             195200205                                                                     LysThrProPheGluLysLysLeuAsnGlyLeuTyrProPheArgVal                              210 215220                                                                    AsnSerAlaGlnArgThrProValGlnMetMetTyrLeuArgGluLys                              225230235240                                                                  LeuAsnIleGlyTyrIleGluAspLeuLysAl aGlnIleLeuGluLeu                             245250255                                                                     ProTyrAlaGlyAspValSerMetPheLeuLeuLeuProAspGluIle                              260265 270                                                                    AlaAspValSerThrGlyLeuGluLeuLeuGluSerGluIleThrTyr                              275280285                                                                     AspLysLeuAsnLysTrpThrSerLysAspLysMetAlaGluAspGlu                              290 295300                                                                    ValGluValTyrIleProGlnPheLysLeuGluGluHisTyrGluLeu                              305310315320                                                                  ArgSerIleLeuArgSerMet GlyMetGluAspAlaPheAsnLysGly                             325330335                                                                     ArgAlaAsnPheSerGlyMetSerGluArgAsnAspLeuPheLeuSer                              340345 350                                                                    GluValPheHisGlnAlaMetValAspValAsnGluGluGlyThrGlu                              355360365                                                                     AlaAlaAlaGlyThrGlyGlyValMetThrGlyArgThrGlyHi sGly                             370375380                                                                     GlyProGlnPheValAlaAspHisProPheLeuPheLeuIleMetHis                              385390395400                                                                  LysIleThr AsnCysIleLeuPhePheGlyArgPheSerSerPro                                405410415                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1512 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      ( D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (G) CELL TYPE: Monocyte                                                       (H) CELL LINE: U937                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: BTA 1916                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 22..1200                                                         (C) IDENTIFICATION METHOD: experimental                                      (D) OTHER INFORMATION: /function="Product binds to                            urokinase, tissue plasminogen activator"                                      /product="PAI-2 variant, protease sensitive site                              removed"                                                                      /evidence=EXPERIMENTAL                                                        /note="Codes for human plasminogen activator                                  inhibitor type 2 protein in which amino acids 74                              to 96 inclusive have been deleted."                                           (x i) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                      GATCTGTAAGGAGGTATATAAATGGAGGATCTTTGTGTGGCAAACACACTC51                         MetGluAspLeuCysValAlaAsnThrLeu                                                15 10                                                                         TTTGCCCTCAATTTATTCAAGCATCTGGCAAAAGCAAGCCCCACCCAG99                            PheAlaLeuAsnLeuPheLysHisLeuAlaLysAlaSerProThrGln                              1520 25                                                                       AACCTCTTCCTCTCCCCATGGAGCATCTCGTCCACCATGGCCATGGTC147                           AsnLeuPheLeuSerProTrpSerIleSerSerThrMetAlaMetVal                              3035 40                                                                       TACATGGGCTCCAGGGGCAGCACCGAAGACCAGATGGCCAAGGTGCTT195                           TyrMetGlySerArgGlySerThrGluAspGlnMetAlaLysValLeu                              4550 55                                                                       CAGTTTAATGAAGTGGGAGCCAATGCAGTTACCCCCATGACTCCAGCA243                           GlnPheAsnGluValGlyAlaAsnAlaValThrProMetThrProAla                              606570                                                                        CAA GCTGCAGATAAAATCCATTCATCCTTCCGCTCTCTCAGCTCTGCA291                          GlnAlaAlaAspLysIleHisSerSerPheArgSerLeuSerSerAla                              75808590                                                                       ATCAATGCATCCACAGGGAATTATTTACTGGAAAGTGTCAATAAGCTG339                          IleAsnAlaSerThrGlyAsnTyrLeuLeuGluSerValAsnLysLeu                              95100105                                                                      TTTGGTGAGAAGTCTGCGAGCTTCCGGGAAGAATATATTCGACTCTGT387                           PheGlyGluLysSerAlaSerPheArgGluGluTyrIleArgLeuCys                              110115120                                                                     C AGAAATATTACTCCTCAGAACCCCAGGCAGTAGACTTCCTAGAATGT435                          GlnLysTyrTyrSerSerGluProGlnAlaValAspPheLeuGluCys                              125130135                                                                     GCAGAA GAAGCTAGAAAAAAGATTAATTCCTGGGTCAAGACTCAAACC483                          AlaGluGluAlaArgLysLysIleAsnSerTrpValLysThrGlnThr                              140145150                                                                     AAAGGCAAAATCCCA AACTTGTTACCTGAAGGTTCTGTAGATGGGGAT531                          LysGlyLysIleProAsnLeuLeuProGluGlySerValAspGlyAsp                              155160165170                                                                  ACCAGGATGGT CCTGGTGAATGCTGTCTACTTCAAAGGAAAGTGGAAA579                          ThrArgMetValLeuValAsnAlaValTyrPheLysGlyLysTrpLys                              175180185                                                                     ACTCCATTTG AGAAGAAACTAAATGGGCTTTATCCTTTCCGTGTAAAC627                          ThrProPheGluLysLysLeuAsnGlyLeuTyrProPheArgValAsn                              190195200                                                                     TCGGCTCAGCGC ACACCTGTACAGATGATGTACTTGCGTGAAAAGCTA675                          SerAlaGlnArgThrProValGlnMetMetTyrLeuArgGluLysLeu                              205210215                                                                     AACATTGGATACATAGAA GACCTAAAGGCTCAGATTCTAGAACTCCCA723                          AsnIleGlyTyrIleGluAspLeuLysAlaGlnIleLeuGluLeuPro                              220225230                                                                     TATGCTGGAGATGTTAGCATGTTCTT GTTGCTTCCAGATGAAATTGCC771                          TyrAlaGlyAspValSerMetPheLeuLeuLeuProAspGluIleAla                              235240245250                                                                  GATGTGTCCACTGGCTTGGAGC TGCTGGAAAGTGAAATAACCTATGAC819                          AspValSerThrGlyLeuGluLeuLeuGluSerGluIleThrTyrAsp                              255260265                                                                     AAACTCAACAAGTGGACCAGC AAAGACAAAATGGCTGAAGATGAAGTT867                          LysLeuAsnLysTrpThrSerLysAspLysMetAlaGluAspGluVal                              270275280                                                                     GAGGTATACATACCCCAGTTCAAA TTAGAAGAGCATTATGAACTCAGA915                          GluValTyrIleProGlnPheLysLeuGluGluHisTyrGluLeuArg                              285290295                                                                     TCCATTCTGAGAAGCATGGGCATGGAGGA CGCCTTCAACAAGGGACGG963                          SerIleLeuArgSerMetGlyMetGluAspAlaPheAsnLysGlyArg                              300305310                                                                     GCCAATTTCTCAGGGATGTCGGAGAGGAATGACCTGT TTCTTTCTGAA1011                         AlaAsnPheSerGlyMetSerGluArgAsnAspLeuPheLeuSerGlu                              315320325330                                                                  GTGTTCCACCAAGCCATGGTGGATGTGAATGAG GAGGGCACTGAAGCA1059                         ValPheHisGlnAlaMetValAspValAsnGluGluGlyThrGluAla                              335340345                                                                     GCCGCTGGCACAGGAGGTGTTATGACAGGGAGA ACTGGACATGGAGGC1107                         AlaAlaGlyThrGlyGlyValMetThrGlyArgThrGlyHisGlyGly                              350355360                                                                     CCACAGTTTGTGGCAGATCATCCTTTTCTTTTTCT TATTATGCATAAG1155                         ProGlnPheValAlaAspHisProPheLeuPheLeuIleMetHisLys                              365370375                                                                     ATAACCAACTGCATTTTATTTTTCGGCAGATTTTCCTCAC CCTAAAACTAAG1207                     IleThrAsnCysIleLeuPhePheGlyArgPheSerSerPro                                    380385390                                                                     CGTGCTGCTTCTGCAAAAGATTTTTGTAGATGAGCTGTGTGCCTCAGAATTGCTATTTCA1267              AATTGCCAAAAATTTAGAGATGTTTTCTACATATTTCTGCTCTTCTGAACAACTTCTGCT1327              ACCCACTAAATAAAAACACAGAAATAATTAGACAATTGTCTATTATAACATGACAACCCT1387              ATTAATCATTTGGTCTTCTAAAATGGGATCATGCCCATTTAGA TTTTCCTTACTATCAGT1447             TTATTTTTATAACATTAACTTTTACTTTGTTATTTATTATTTTATATAATGGTGAGTTTT1507              TGGGG1512                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 392 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetGluAspLeuCysValAlaAsnThrLeuPheAlaLeuAsnLeuPhe                              1510 15                                                                       LysHisLeuAlaLysAlaSerProThrGlnAsnLeuPheLeuSerPro                              202530                                                                        TrpSerIleSerSerThrMetAlaMetValTyrMetGlySerArgGly                              354045                                                                        SerThrGluAspGlnMetAlaLysValLeuGlnPheAsnGluValGly                              505560                                                                        AlaAsnAlaValThrProMetT hrProAlaGlnAlaAlaAspLysIle                             65707580                                                                      HisSerSerPheArgSerLeuSerSerAlaIleAsnAlaSerThrGly                              85 9095                                                                       AsnTyrLeuLeuGluSerValAsnLysLeuPheGlyGluLysSerAla                              100105110                                                                     SerPheArgGluGluTyrIleArgLeuCysGlnLys TyrTyrSerSer                             115120125                                                                     GluProGlnAlaValAspPheLeuGluCysAlaGluGluAlaArgLys                              130135140                                                                     LysIleAsnSe rTrpValLysThrGlnThrLysGlyLysIleProAsn                             145150155160                                                                  LeuLeuProGluGlySerValAspGlyAspThrArgMetValLeuVal                              16 5170175                                                                    AsnAlaValTyrPheLysGlyLysTrpLysThrProPheGluLysLys                              180185190                                                                     LeuAsnGlyLeuTyrProPheArgV alAsnSerAlaGlnArgThrPro                             195200205                                                                     ValGlnMetMetTyrLeuArgGluLysLeuAsnIleGlyTyrIleGlu                              210215220                                                                      AspLeuLysAlaGlnIleLeuGluLeuProTyrAlaGlyAspValSer                             225230235240                                                                  MetPheLeuLeuLeuProAspGluIleAlaAspValSerThrGlyLeu                               245250255                                                                    GluLeuLeuGluSerGluIleThrTyrAspLysLeuAsnLysTrpThr                              260265270                                                                     SerLysAspLysMe tAlaGluAspGluValGluValTyrIleProGln                             275280285                                                                     PheLysLeuGluGluHisTyrGluLeuArgSerIleLeuArgSerMet                              290295 300                                                                    GlyMetGluAspAlaPheAsnLysGlyArgAlaAsnPheSerGlyMet                              305310315320                                                                  SerGluArgAsnAspLeuPheLeuSerGluValPheHisG lnAlaMet                             325330335                                                                     ValAspValAsnGluGluGlyThrGluAlaAlaAlaGlyThrGlyGly                              340345350                                                                     Val MetThrGlyArgThrGlyHisGlyGlyProGlnPheValAlaAsp                             355360365                                                                     HisProPheLeuPheLeuIleMetHisLysIleThrAsnCysIleLeu                              370 375380                                                                    PhePheGlyArgPheSerSerPro                                                      385390                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1482 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (G) CELL TYPE: Monocyte                                                       (H) CELL LINE: U937                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: BTA 1922                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 22..1170                                                        (C) IDENTIFICATION METHOD: experimental                                       ( D) OTHER INFORMATION: /function="Product binds to                           urokinase, tissue plasminogen activator"                                      /product="PAI-2 variant, protease sensitive site                              removed"                                                                      /evidence=EXPERIMENTAL                                                        /note="Codes for human plasminogen activator                                  inhibitor type 2 protein in which amino acids 66                              to 98 inclusive have been deleted."                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GATCTGTAAG GAGGTATATAAATGGAGGATCTTTGTGTGGCAAACACACTC51                        MetGluAspLeuCysValAlaAsnThrLeu                                                1510                                                                          TTTG CCCTCAATTTATTCAAGCATCTGGCAAAAGCAAGCCCCACCCAG99                           PheAlaLeuAsnLeuPheLysHisLeuAlaLysAlaSerProThrGln                              152025                                                                        AAC CTCTTCCTCTCCCCATGGAGCATCTCGTCCACCATGGCCATGGTC147                          AsnLeuPheLeuSerProTrpSerIleSerSerThrMetAlaMetVal                              303540                                                                        TACATG GGCTCCAGGGGCAGCACCGAAGACCAGATGGCCAAGGTGCTT195                          TyrMetGlySerArgGlySerThrGluAspGlnMetAlaLysValLeu                              455055                                                                        CAGTTTAATGA AGTGGGAGCCGCTGCAGATAAAATCCATTCATCCTTC243                          GlnPheAsnGluValGlyAlaAlaAlaAspLysIleHisSerSerPhe                              606570                                                                        CGCTCTCTCAGCTCTGCAA TCAATGCATCCACAGGGAATTATTTACTG291                          ArgSerLeuSerSerAlaIleAsnAlaSerThrGlyAsnTyrLeuLeu                              75808590                                                                      GAAAGTGTCAATAAG CTGTTTGGTGAGAAGTCTGCGAGCTTCCGGGAA339                          GluSerValAsnLysLeuPheGlyGluLysSerAlaSerPheArgGlu                              95100105                                                                      GAATATATTCGACTC TGTCAGAAATATTACTCCTCAGAACCCCAGGCA387                          GluTyrIleArgLeuCysGlnLysTyrTyrSerSerGluProGlnAla                              110115120                                                                     GTAGACTTCCTAGAATG TGCAGAAGAAGCTAGAAAAAAGATTAATTCC435                          ValAspPheLeuGluCysAlaGluGluAlaArgLysLysIleAsnSer                              125130135                                                                     TGGGTCAAGACTCAAACCAAAG GCAAAATCCCAAACTTGTTACCTGAA483                          TrpValLysThrGlnThrLysGlyLysIleProAsnLeuLeuProGlu                              140145150                                                                     GGTTCTGTAGATGGGGATACCAGGATGGTC CTGGTGAATGCTGTCTAC531                          GlySerValAspGlyAspThrArgMetValLeuValAsnAlaValTyr                              155160165170                                                                  TTCAAAGGAAAGTGGAAAACTCCATTT GAGAAGAAACTAAATGGGCTT579                          PheLysGlyLysTrpLysThrProPheGluLysLysLeuAsnGlyLeu                              175180185                                                                     TATCCTTTCCGTGTAAACTCGGCTCA GCGCACACCTGTACAGATGATG627                          TyrProPheArgValAsnSerAlaGlnArgThrProValGlnMetMet                              190195200                                                                     TACTTGCGTGAAAAGCTAAACATTGGAT ACATAGAAGACCTAAAGGCT675                          TyrLeuArgGluLysLeuAsnIleGlyTyrIleGluAspLeuLysAla                              205210215                                                                     CAGATTCTAGAACTCCCATATGCTGGAGATGTT AGCATGTTCTTGTTG723                          GlnIleLeuGluLeuProTyrAlaGlyAspValSerMetPheLeuLeu                              220225230                                                                     CTTCCAGATGAAATTGCCGATGTGTCCACTGGCTTGGAGCTG CTGGAA771                          LeuProAspGluIleAlaAspValSerThrGlyLeuGluLeuLeuGlu                              235240245250                                                                  AGTGAAATAACCTATGACAAACTCAACAAGTGGACCAG CAAAGACAAA819                          SerGluIleThrTyrAspLysLeuAsnLysTrpThrSerLysAspLys                              255260265                                                                     ATGGCTGAAGATGAAGTTGAGGTATACATACCCCAGT TCAAATTAGAA867                          MetAlaGluAspGluValGluValTyrIleProGlnPheLysLeuGlu                              270275280                                                                     GAGCATTATGAACTCAGATCCATTCTGAGAAGCATGGGC ATGGAGGAC915                          GluHisTyrGluLeuArgSerIleLeuArgSerMetGlyMetGluAsp                              285290295                                                                     GCCTTCAACAAGGGACGGGCCAATTTCTCAGGGATGTCGGAGAGG AAT963                          AlaPheAsnLysGlyArgAlaAsnPheSerGlyMetSerGluArgAsn                              300305310                                                                     GACCTGTTTCTTTCTGAAGTGTTCCACCAAGCCATGGTGGATGTGAAT10 11                         AspLeuPheLeuSerGluValPheHisGlnAlaMetValAspValAsn                              315320325330                                                                  GAGGAGGGCACTGAAGCAGCCGCTGGCACAGGAGGTGTTATGACAGGG 1059                         GluGluGlyThrGluAlaAlaAlaGlyThrGlyGlyValMetThrGly                              335340345                                                                     AGAACTGGACATGGAGGCCCACAGTTTGTGGCAGATCATCCTTTTCTT 1107                         ArgThrGlyHisGlyGlyProGlnPheValAlaAspHisProPheLeu                              350355360                                                                     TTTCTTATTATGCATAAGATAACCAACTGCATTTTATTTTTCGGCAGA 1155                         PheLeuIleMetHisLysIleThrAsnCysIleLeuPhePheGlyArg                              365370375                                                                     TTTTCCTCACCCTAAAACTAAGCGTGCTGCTTCTGCAAAAGATTTTTGTAGA1207                       PheSerSerPro                                                                 380                                                                           TGAGCTGTGTGCCTCAGAATTGCTATTTCAAATTGCCAAAAATTTAGAGATGTTTTCTAC1267              ATATTTCTGCTCTTCTGAACAACTTCTGCTACCCACTAAATAAAAACACAGAAATAATTA1327              GACAATTGTCTATTATAACAT GACAACCCTATTAATCATTTGGTCTTCTAAAATGGGATC1387             ATGCCCATTTAGATTTTCCTTACTATCAGTTTATTTTTATAACATTAACTTTTACTTTGT1447              TATTTATTATTTTATATAATGGTGAGTTTTTGGGG1482                                       (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 382 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetGluAspLeuCysValAlaAsnThrLeuPheAlaLeuAsnLeuPhe                              15 1015                                                                       LysHisLeuAlaLysAlaSerProThrGlnAsnLeuPheLeuSerPro                              202530                                                                        TrpSerIleSerSerThrMetAlaMetVal TyrMetGlySerArgGly                             354045                                                                        SerThrGluAspGlnMetAlaLysValLeuGlnPheAsnGluValGly                              505560                                                                        AlaA laAlaAspLysIleHisSerSerPheArgSerLeuSerSerAla                             65707580                                                                      IleAsnAlaSerThrGlyAsnTyrLeuLeuGluSerValAsnLysLeu                               859095                                                                       PheGlyGluLysSerAlaSerPheArgGluGluTyrIleArgLeuCys                              100105110                                                                     GlnLysTyrTyrSerSer GluProGlnAlaValAspPheLeuGluCys                             115120125                                                                     AlaGluGluAlaArgLysLysIleAsnSerTrpValLysThrGlnThr                              130135 140                                                                    LysGlyLysIleProAsnLeuLeuProGluGlySerValAspGlyAsp                              145150155160                                                                  ThrArgMetValLeuValAsnAlaValTyrPheLysGlyLysTrp Lys                             165170175                                                                     ThrProPheGluLysLysLeuAsnGlyLeuTyrProPheArgValAsn                              180185190                                                                     SerAlaG lnArgThrProValGlnMetMetTyrLeuArgGluLysLeu                             195200205                                                                     AsnIleGlyTyrIleGluAspLeuLysAlaGlnIleLeuGluLeuPro                              2102 15220                                                                    TyrAlaGlyAspValSerMetPheLeuLeuLeuProAspGluIleAla                              225230235240                                                                  AspValSerThrGlyLeuGluLeuLeuGluSer GluIleThrTyrAsp                             245250255                                                                     LysLeuAsnLysTrpThrSerLysAspLysMetAlaGluAspGluVal                              260265 270                                                                    GluValTyrIleProGlnPheLysLeuGluGluHisTyrGluLeuArg                              275280285                                                                     SerIleLeuArgSerMetGlyMetGluAspAlaPheAsnLysGlyArg                              290 295300                                                                    AlaAsnPheSerGlyMetSerGluArgAsnAspLeuPheLeuSerGlu                              305310315320                                                                  ValPheHisGlnAlaMetValA spValAsnGluGluGlyThrGluAla                             325330335                                                                     AlaAlaGlyThrGlyGlyValMetThrGlyArgThrGlyHisGlyGly                              340345 350                                                                    ProGlnPheValAlaAspHisProPheLeuPheLeuIleMetHisLys                              355360365                                                                     IleThrAsnCysIleLeuPhePheGlyArgPheSerSerPro                                     370375380                                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide                                (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGCCCATATGATATCTCGAGACTAGTC27                                                 (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA adaptor for replacing HinfI/Pst                region of PAI-2 gene in 74-96 amino acid coding                               region deletion variant.                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..18                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ACTCCAGCACAAGCTGCA 18                                                         ThrProAlaGlnAlaAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ThrPro AlaGlnAlaAla                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Complementary sequence to SEQ ID NO:8 adaptor                for replacing HinfI/PstI region of PAI-2 gene in                               74-96 amino acid coding region deletion variant.                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCTTGTGCTGG11                                                                 (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide for use in PCR                 reaction to create gene encoding 66-98 amino                                  acid deletion variant of PAI-2.                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CCTCTTCTGCAGATTCTAGGAA 22                                                     (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide for use in PCR                 reaction to create gene encoding 66-98 amino                                   acid deletion variant of PAI-2.                                              (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ATCTGCAGCGGCTCCCACTTCATTAAACT29                                               (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide for use in PCR                 reaction to create gene encoding 66-98 amnio                                  acid deletion variant of PAI-2.                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GTGGGAGCCGCTGCAGATAAAATCCATT 28                                               (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide                                (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GATCTNNNNNNNNNNNNNNNNATGGAG27                                                 (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                        (A) DESCRIPTION: Synthetic DNA oligonucleotide                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GATCTNNNNNNNNNNNNNNNATGGAG26                                                  (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      LysGlySerTyrProAspAlaIleLeuGlnAlaGlnAlaAlaAsp                                 1510 15                                                                       (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      PheMetGlnGlnIleGlnLysGlySe rTyr                                               1510                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GlyPheMetGlnGlnIleGlnLysGlySerTyrProAspAlaIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide                                (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..27                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CAGCAGATCCAGGCAGGTAGTTATCCT 27                                                GlnGlnIleGlnAlaGlySerTyrPro                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GlnG lnIleGlnAlaGlySerTyrPro                                                  15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide, complement to                  SEQ ID NO:19.                                                                (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AGGATAACTACCTGCCTGGATCTGCTG27                                             